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Split focal adhesion kinase for probing protein–protein interactions

Paper ID Volume ID Publish Year Pages File Format Full-Text
3017 147 2014 7 PDF Available
Title
Split focal adhesion kinase for probing protein–protein interactions
Abstract

•We propose a novel system to detect protein–protein interaction in mammalian cells.•The distinct domains in focal adhesion kinase are tethered to two target proteins.•Interaction between target proteins leads to phosphorylation at specific tyrosines.•FKBP–FRB interaction was clearly detected by probing the phosphorylation.

Since protein–protein interactions (PPIs) regulate a variety of cellular processes, the detection of PPIs is crucial for elucidating the underlying molecular mechanisms as well as developing therapeutics. In this study, we propose a novel system to detect PPIs using the distinct domains of focal adhesion kinase (FAK). In this system named “split FAK”, the linker and kinase domains in native FAK are tethered separately to two target proteins of interest. The interaction between the target proteins brings the linker and kinase domains into proximity, which leads to phosphorylation at Y397 of the linker domain, recruitment of another tyrosine kinase Src, and phosphorylation at Y576 of the kinase domain. PPIs are readily detected by probing phosphorylation at Y397 and Y576 of these domains. To demonstrate this system, we designed a series of split FAK chimeras with different domain structures. Consequently, dimerizer-induced interaction between FK506-binding protein 12 (FKBP) and the T2098L mutant of FKBP12-rapamycin binding domain (FRB) was clearly detected by probing phosphorylation at the specific tyrosine residues of most of the split FAK chimeras. This is a novel PPI detection system based on a mechanism-inspired design of a trans-activated split kinase.

Keywords
Protein–protein interaction; Focal adhesion kinase; Animal cell culture; Biomedical; Protein; Recombinant DNA
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Split focal adhesion kinase for probing protein–protein interactions
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 90, 15 September 2014, Pages 272–278
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us